The growth and maintenance of cells derived from animal and human tissues and the establishment of cell lines are important for many different purposes. For example, tissue culture is used extensively for the production of enzymes, cell products such as the lymphokines, plasminogen activator, antibodies etc., and for the general testing of drugs, carcinogenic and chemotherapeutic agents and the like, and in studies of infectious diseases, cancerous diseases, and inherited or acquired disorders. With the development of cell fusion and the preparation of monoclonal antibodies, a directed emphasis has been placed on in vitro growth of animal and human cell lines for the development of diagnostic tests. Cultured human cells themselves are being used for the therapy of cancer patients by various kinds of adoptive immunity.
It is well-known that specific cell lines can be grown in vitro in optimally formulated culture or nutrient media (Hanss et al. (1964) Exp. Cell Res. 34:243-256; Fahey et al. (1966) Science 152:1259-1261; and Ham and McKeehan (1979) Methods in Enzymol. 58:44-93). Some examples of culture media developed for special purposes are: RPMI 1640 medium for optimal growth of human B-lymphoid cells (Moore et al. (1967) J. Am. Med. Assoc. 199:519-524) and diverse kinds of malignant cells, Changs medium for optimal growth of amniotic fluid cells, medium 199 for optimal growth of mouse fibroblast cells, MEM medium, a "minimal" medium for optimal growth of attached mammalian cells, Leibovitz medium for optimal growth in absence of CO.sub.2, F10 medium for optimal growth of liver cells, etc. Such media are distinguished from one another in that they contain critically different components in precise amounts. These components include amino acids, vitamins, inorganic salts, trace elements and other organic compounds which promote the maximum growth of the cultured cells. For most purposes media are supplemented with serum.
The growth of various mammalian cells in vitro has been achieved in several chemically defined media supplemented with various sera, preferably fetal calf or newborn calf serum and other incompletely defined growth factors. Unfortunately, the addition of serum, whose constituents may vary widely, introduces undefined biological components into the nutrient medium and, hence, contributes to the variability of biochemical and cellular events. Furthermore, serum is expensive and in some instances, has been shown to be inhibitory to the growth of certain cells and may result in critical immune reactions in patients if the cells are used for clinical purposes.
The possibility of growing certain types of cells in serum-free medium was undertaken by several laboratories, e.g., Sato (1975) in Biochemical Actions of Hormones, G. Litwack (ed.), Academic Press, New York, Vol. 3, pp. 391-396; Bottenstein et al. (1978) Methods in Enzymol. 58:94-108; Beebe et al. (1987) Cancer Res. 47:2380-2384; Cole et al. (1987) J. Immunol. Meth. 97:29-35; Ham et al. (August 1988) In Vitro 24:833-844). Replacement of serum with supplements of better defined composition was found to be not very successful for the growth of normal cells in conventional culture media, although in vitro growth of some kinds of malignant cells was supported under these conditions after a period of adaptation. Only relatively slow growth of a selected few kinds of cells was possible in the absence of supplemental proteins.
Gradually, the amount of undefined supplementation needed for good growth was reduced by a process of optimization in particular basal nutrient media for selected cell lines (Mizrahi et al. (1970) Appl. Microbiol. 19:906-10; Ham (1984) in Methods for Preparation of Media, Supplements and Substrata for Serum-Free Animal Cell Culture, Alan R. Liss, Inc., New York, pp. 3-21). Moore and Woods (1977) Tissue Culture Assoc. Manual 3:503-509 expressed the generally held tenet in the art that "Some media will be better for initiating cultures of fresh cells while others will be better suited for maintaining established cell lines. Media that provide vigorous cell reproduction may suppress cell differentiation." These authors, like others in the field believed that the elements of a successful culture medium for mammalian cells were determined by the kind of cells to be cultured, unknown nutritional supplements, osmolarity, buffers and pH levels, gaseous mixtures and other environmental aspects of the culture unit. Thus, emphasis was placed on development of optimized formulation of culture media for specific purposes and for selected cell lines as well as for the formulation of general purpose media.
In the 1960's the RPMI 1640 culture medium was formulated (Moore et al. (1967) supra). This medium is still the most widely used today for culturing human normal and neoplastic B-lymphoid cells, malignant human cells and many other kinds of mammalian cells. RPMI 1640 supplemented with 10 to 20% heat-inactivated fetal bovine serum (FBS) is proficient for initiating many types of cell cultures, and maintaining established cell lines when supplemented with 10 or 5% FBS. These media were shown to be unusually good for culturing fresh human lymphocytes and for supporting cell cultures derived from mice, rats and hamsters (Moore and Woods (1977) supra). Subsequently, several other media, e.g., GEM 1717, COF 1755, COF 1759, COF 1767, etc. were formulated by Applicant. Each of these media differs from RPMI 1640 with respect to composition, concentration of components and final ratios among individual components. A number of these media are sold commercially and are widely used for experimental and long-term tissue cultures (see catalogs from commercial vendors of scientific and biological products, e.g., Sigma Chemical Company, St. Louis, Mo.; Gibco, Grand Island, N.Y.; Irvine Scientific, Santa Ana, Calif.; J.R. Scientific, Woodland, Calif.; Hazeton Biologics, Lenexa, Kans.; etc.).
Simms et al. (1980) Cancer Res. 40: 4356-4363 studied the supplemental growth factor requirements for replication of human small cell carcinomas of the lung in serum-free RPMI 1640 medium, and developed a formula containing selenium, hydrocortisone, insulin, transferrin and 17.beta.-estradiol (known as Hites). The Hites combination added to RPMI 1640 medium devoid of serum supplementation supported optimum replication of cultured lung carcinoma cells. The cells replicated with approximately the same doubling times as in RPMI 1640 medium supplemented with 10% fetal calf serum but they exhibited a lower cell population density and a longer lag phase in Hites-supplemented medium.
RPMI 1640 was also the basis for other formulae of media to be used without serum supplementation. Darbre et al. (1984) Cancer Res. 44:2790-2793 utilized serum-free RPMI 1640 medium supplemented with penicillin, streptomycin, HEPES buffer, insulin, hydrocortisone, transferrin, 3,3',5-triiodo-L-thyronine, epidermal growth factor (EGF), fibroblast growth factor (FGF) and fibronectin to grow human mammary cancer cells in monolayer and suspension culture to test and compare the effects of estradiol and tamoxifen. The growth rate of the mammary cancer cells in this serum-free medium was adequate but slower than in medium supplemented with serum.
A serum-free medium was developed by Ohmori (1988) J. Immunol. Methods 112:227-233, which supported primary antibody responses by cultured murine lymphocytes. The medium used was RPMI 1640 supplemented with .beta.-cyclodextrin, insulin, transferrin, albumin, low density lipoprotein, putrescine and L-alanine as substitutes for fetal serum. This serum-free medium supported the antibody response to sheep erythrocytes, trinitrophenyl-Ficoll or trinitrophenyl-lipopolysaccharide as efficiently as 10% fetal calf serum containing medium.
O'Donnell-Tormey et al. (1987) J. Exp. Med. 165:500-514 studied the metabolism of pyruvate in normal and malignant mammalian cells grown and maintained in serum-free RPMI 1640 supplemented with nonessential amino acids, penicillin, streptomycin and additional glutamine. Their work suggested that pyruvate and related .alpha.-ketoacids protect mammalian cells in culture against peroxide-induced cytotoxicity.
In vitro lymphocyte proliferation, primarily of B-cells, was shown to be optimal when RPMI 1640 culture medium was supplemented with 5 to 20% serum. Soyano et al. (1985) Immunol. Lett. 9:57-62 were able to decrease the level of serum added to RPMI 1640 medium to as low as 1% and still obtain an adequate proliferation of human peripheral blood lymphocytes. They further found that in serum-free cultures, wherein RPMI 1640 is supplemented with additional glutamine, penicillin, streptomycin, sodium bicarbonate, transferrin and albumin, the proliferation rate of lymphocytes was approximately 80% of that obtained in the presence of 1% human serum. These authors further observed that albumin and transferrin acted synergistically on the mitogen stimulation of human mononuclear leukocytes.